A biosensor, which utilizes a specific reaction produced by an antibody, an enzyme and the like, detects components to be measured in an inspection target solution such as body fluid, thereby enabling application to a clinical field or the like.
As a typical example of qualitatively or quantitatively measuring components to be measured in an inspection target solution (sample) which is detected by such a biosensor, utilizing chromatography, there is an immuno-chromatographic sensor. In this immuno-chromatographic sensor, a development layer for developing the inspection target solution is provided, which includes a reagent part where a reagent is immobilized in a part thereof, and also includes a marker reagent hold part where a marker reagent, which can be eluted by development of the inspection target solution, is held in a dry state. When the a inspection target solution is developed, bonding occurs in the reagent immobilization part and then this bonding amount is measured, whereby components to be measured in the inspection target solution can be measured.
This immuno-chromatographic sensor is generally constituted by an inspection target solution application part where an inspection target solution is applied, and plural development layers, in which an antibody is immobilized in a reagent immobilization part, which is a part of the development layer, and an antibody marked with a marker such as gold colloid particles is held, in a marker reagent hold part which is upstream of the reagent immobilization part in the development layer, in a dry state where it can be eluted by the inspection target solution.
When a required amount of inspection target solution is applied to this immuno-chromatographic sensor, the inspection target solution elutes the marked antibody while permeating porous materials. Then, an amount of the marked antibody, which is the amount of the marked antibody eluted by the inspection target solution being bonded to the antibody in the reagent immobilization part, in the reagent immobilization part is measured, thereby detecting components to be measured in the inspection target solution. Further, the amount of this bonded marked antibody can be measured by visually confirming the amount of the marker, such as gold colloid particles which remains behind as a result of the bonding of the marked antibody in the reagent immobilization part. That is, since the degree of coloration (the depth of a color) in the reagent immobilization part varies with the concentration of an antigen (components to be measured) included in the inspection target solution, the inspector visually checks this, thereby enabling the measurement.
While the description has been given here of a case where a sandwich reaction of an antigen antibody reaction is taken as a measurement principle, the measurement result can also be obtained in a bonding state of the marker reagent in an antibody immobilization part even when other competitive reactions are similarly taken as measurement principles.
Further, while in the above-described example the description is given of a case where the measurement result is visually obtained by a qualitative judgement, Japanese Published Patent Application No. Hei. 8-334511 describes a method by which the degree of coloration in the reagent immobilization part of a specimen is imaged by a CCD to be judged automatically so as to improve a low reproducibility and individual variations of the visual judgement. In a case where a semi-quantitative or more accurate judgement is required as the measurement result, there are a method for reading by a transparent mode employing an optical reading device, which is disclosed in Japanese Published Patent Application No. Hei. 10-274624, and a method for taking-in as an image by a camera or the like to be processed arithmetically, which is disclosed in Japanese Published Patent Application No. Hei. 10-274653.
However, in the above-described conventional methods for measuring a measurement target in an inspection target solution, an elution amount of the marked antibody eluted from the marker reagent hold part by the inspection target solution varies due to factors coming from a dry state of a sensor, a preservation condition of a reagent in the sensor, or an error in reagent concentration in the sensor depending on environments at fabrication of the sensor, factors coming from a property of the inspection target solution, factors coming from an erroneous operation at measurement or environments at the measurement, or the like.
When the elution amount of the marked antibody is constant, the amount of the bonded marked antibody which is obtained by the marked antibody being eluted into the reagent immobilization part and thereby bonded enables more accurate and uniform measurement of the concentration of the measurement target in the inspection target solution. However, when the amount of the eluted marked antibody varies due to the above-described factors, the amount of the marked antibody which is bonded to the reagent immobilization part for each measurement will vary even when the concentration of the measurement target in the inspection target solution is constant.
That is, in a case where the amount of the eluted marked antibody varies due to the factors when the measurement is implemented to obtain a qualitative measurement result, an erroneous measurement result is given especially in the vicinity of a measurement limit high sensitivity.
Further, when the amount of the eluted marked antibody is extremely small, an accurate bonding amount cannot be obtained in the reagent immobilization part.
Moreover, also in the case of implementing a measurement requiring a semi-quantitative or quantitative measurement, the bonding amount of the marked antibody in the reagent immobilization part varies due to variations in the amount of the eluted marked antibody due to the factors, resulting only in a low-accurate measurement. Thus, the above-described methods can only implement at most a qualitative or semi-quantitative measurement at the most and cannot realize a quantitative measurement.
The present invention is made to solve the above-mentioned problems and has for its object to provide a chromatography measuring method which can obtain a more accurate measurement result and enhance its accuracy also of a quantitative measurement.